epidermal-growth-factor and Osteosarcoma

Topics: Animals; Antigens, Viral; Cell Transformation, Neoplastic; Cricetinae; Epidermal Growth Factor; Humans; Laryngeal Neoplasms; Mice; Neoplasms, Radiation-Induced; Oncogenes; Oncogenic Viruses; Osteosarcoma; Peptides; Repetitive Sequences, Nucleic Acid; Transforming Growth Factors

Kallmann syndrome is the result of innate genetic defects in the fibroblast growth factor (FGF) regulated signaling network causing diminished signal transduction. One of the rare mutations associated with the syndrome alters the Sprouty (Spry)4 protein by converting the serine at position 241 into a tyrosine. In this study, we characterize the tyrosine Spry4 mutant protein in the primary human embryonic lung fibroblasts WI-38 and osteosarcoma-derived cell line U2OS. As demonstrated in a cell signaling assay, Spry4 gains the capability of inhibiting FGF, but not epithelial growth factor (EGF)-induced signaling as a consequence of the tyrosine substitution. Additionally, migration of normal embryonic lung fibroblasts and osteosarcoma-derived cells is potently inhibited by the tyrosine Spry4 variant, while an effect of the wildtype Spry4 protein is hardly measureable. Concerning cell proliferation, the unaltered Spry4 protein is ineffective to influence the WI-38 cells, while the mutated Spry4 protein decelerates the cell doubling. In summary, these data emphasize that like the other mutations associated with Kallmann syndrome the described Spry4 mutation creates a hyperactive version of a selective inhibitory molecule and can thereby contribute to a weakened FGF signaling. Additionally, the study pinpoints a Spry4 variation expanding the applicability of Spry4 in a potential cancer therapy.

Topics: Cell Line; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epidermal Growth Factor; Fibroblast Growth Factor 2; Fibroblasts; Humans; Intracellular Signaling Peptides and Proteins; Kallmann Syndrome; Lung; MAP Kinase Signaling System; Mutation; Nerve Tissue Proteins; Osteosarcoma; Phosphorylation; Signal Transduction; Tyrosine

BACKGROUND The present work was performed to detect the potential inhibitory effect of cyclic guanosine monophosphate (cGMP)-dependent protein kinase II (PKG II) on epidermal growth factor (EGF) receptor-induced biological activity and related signal cascades in osteosarcoma cells. MATERIAL AND METHODS We transfected the osteosarcoma MG-63 cell line with an adenoviral vector encoding PKG II cDNA (Ad-PKGII) and incubated the transfected cells with 250 μM 8-pCPT-cGMP to activate the PKG II. We stimulated the MG-63 cells with100 ng/ml EGF, and then detected their proliferation using a CCK-8 assay. Transwell assay was used to examine MG-63 cell migration; and Western blot analysis was used to detect expression of matrix metalloproteinase 9 (MMP-9) and activation of ERK and Akt. RESULTS Stimulating cells by 100 ng/ml EGF promoted MG-63 cell proliferation and migration, ERK and Akt phosphorylation, and MMP-9 expression. These effects of EGF were inhibited in MG-63 cells infected with Ad-PKGII and incubated with 8-pCPT-cGMP. CONCLUSIONS Our results demonstrate that Ad-PKGII infection significantly inhibited EGF-induced proliferation and migration, as well as the associated-signal cascades; which indicates that PKG II might be a potential anti-cancer factor.

Topics: Bone Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclic GMP; Cyclic GMP-Dependent Protein Kinase Type II; Epidermal Growth Factor; ErbB Receptors; Humans; Osteosarcoma; Phosphorylation; Protein Binding; Signal Transduction; Transfection

BACKGROUND Epithelial-mesenchymal transition (EMT) is responsible for metastasis of cancers, and NF-κB can promote tumor progression. Ezrin is an important molecule participating in EMT. However, whether Ezrin mediates NF-κB in EGF-induced osteosarcoma is unknown. MATERIAL AND METHODS Ezrin phosphorylation, NF-κB activation, and EGF-induced EMT were studied in MG63 and U20S cells with NF-κB inhibition, silencing, or over-expressing Ezrin. Cell morphology, proliferation, migration, and motility were analyzed. An osteosarcoma model was established in mice by injecting MG63 and U20S and reducing Ezrin. RESULTS With EGF induction in vitro, Ezrin Tyr353 and Thr567 were phosphorylated, and EMT, proliferation, migration, and motility of osteosarcoma cells were promoted. Silencing Ezrin suppressed and over-expressing Ezrin promoted the nuclear translocation of p65 and phosphorylated IκBα (p-IκBα) in EGF-induced osteosarcoma cells. NF-κB inhibitor blocked EGF-induced EMT in both cell types, as well as reserving cell morphology and suppressing proliferation, migration, and motility. In vivo, reducing Ezrin significantly suppressed metastasis of osteosarcoma xenografts, increased liver and lung weights, and activated NF-κB, which were both induced by EGF. CONCLUSIONS Ezrin/NF-κB regulated EGF-induced EMT, as well as progression and metastasis of osteosarcoma in vivo and in vitro. Ezrin/NF-κB may be a new therapeutic target to prevent osteosarcoma from deterioration.

Topics: Animals; Bone Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cytoskeletal Proteins; Disease Models, Animal; Disease Progression; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; Heterografts; Male; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; NF-kappa B; NF-KappaB Inhibitor alpha; Osteosarcoma; Phosphorylation; Signal Transduction

Osteosarcoma, the most common primary bone tumor, occurs most frequently in children and adolescents and has a 5-year survival rate, which is unsatisfactory. As epidermal growth factor receptor (EGFR) positively correlates with TNM (tumor-node-metastasis) stage in osteosarcoma, EGFR may play an important role in its progression. The purpose of this study was to explore potential mechanisms underlying this correlation. We found that EGF promotes MG63 cell migration and invasion as well as stress fiber formation via Rho A activation and that these effects can be reversed by inhibiting Rho A expression. In addition, molecules downstream of Rho A, including ROCK1, LIMK2, and Cofilin, are activated by EGF in MG63 cells, leading to actin stress fiber formation and cell migration. Moreover, inhibition of ROCK1, LIMK2, or Cofilin in MG63 cells using known inhibitors or short hairpin RNA (shRNA) prevents actin stress fiber formation and cell migration. Thus, we conclude that Rho A/ROCK1/LIMK2/Cofilin signaling mediates actin microfilament formation in MG63 cells upon EGFR activation. This novel pathway provides a promising target for preventing osteosarcoma progression and for treating this cancer.

Topics: Bone Neoplasms; Cell Line, Tumor; Cell Movement; Cofilin 1; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Neoplastic; Humans; Lim Kinases; Osteosarcoma; rho-Associated Kinases; rhoA GTP-Binding Protein; Signal Transduction; Stress Fibers

Mammalian mitochondria can be transferred between cells both in culture and in vivo. There is evidence that isolated mitochondria enter cells by endocytosis, but the mechanism has not been fully characterised. We investigated the entry mechanism of isolated mitochondria into human osteosarcoma (HOS) cells. Initially we confirmed that respiratory-competent cells can be produced following incubation of HOS cells lacking mitochondrial DNA (mtDNA) with functional exogenous mitochondria and selection in a restrictive medium. Treatment of HOS cells with inhibitors of different endocytic pathways suggest that uptake of EGFP-labelled mitochondria occurs via an actin-dependent endocytic pathway which is consistent with macropinocytosis. We later utilised time-lapse microscopy to show that internalised mitochondria were found in large, motile cellular vesicles. Finally, we used confocal imaging to show that EGFP-labelled mitochondria colocalise with a macropinocytic cargo molecule during internalisation, HOS cells produce membrane ruffles interacting with external mitochondria during uptake and EGFP-labelled mitochondria are found within early macropinosomes inside cells. In conclusion our results are consistent with isolated mitochondria being internalised by macropinocytosis in HOS cells.

Topics: Base Sequence; Cell Line, Tumor; DNA, Mitochondrial; Epidermal Growth Factor; HEK293 Cells; Humans; Mitochondria; Osteosarcoma; Oxidative Phosphorylation; Pinocytosis; Transport Vesicles

The molecular regulation of the invasion of osteosarcoma (OS) remains elusive. Here, we reported significant lower level of miR-143 and significant levels of phosphorylated EGFR and MMP9 in the resected OS from the patients, compared to the adjacent normal tissue. Moreover, strong correlation was detected among these three factors. We thus hypothesized existence of a causal link, which prompted us to use two human OS cell lines to study the interaction of miR-143, MMP9, and activation of EGFR signaling. We found that EGF-induced EGFR phosphorylation in both lines activated MMP9, and consequently cancer invasiveness. Both an inhibitor for EGFR phosphorylation and an inhibitor for ERK1/2 phosphorylation significantly inhibited the EGF-induced activation of MMP9. Moreover, miR-143 levels did not alter by EGF-induced EGFR phosphorylation, while overexpression of miR-143 antagonized EGF-induced MMP9 activation without affecting EGFR phosphorylation. Taken together, our data suggest that miR-143 inhibits EGFR signaling through its downstream ERK/MAPK signaling cascades to control MMP9 expression in OS. Thus, miR-143, EGFR, and MMP9 are therapeutic targets for inhibiting OS invasion.

Topics: Bone Neoplasms; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Gene Expression; Gene Expression Regulation, Neoplastic; Humans; MAP Kinase Signaling System; Matrix Metalloproteinase 9; MicroRNAs; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neoplasm Invasiveness; Osteosarcoma; Phosphorylation; Signal Transduction

To explore the effects of a knockdown of cyclooxygenase 2 upon the growth and migration of SaOS2 cells.. We employed lentivirus mediated-RNA interference technology to knockdown the endogenous expression of gene COX-2 in human osteosarcoma cells (SaOS2) and analyzed their phenotypical changes. The effects of COX-2 silencing on the proliferation, cell cycle and migration of SaOS2 cells were assessed by thiazolyl blue tetrazolium bromide, flow cytometry and migration assays respectively. COX-2, vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) mRNA and protein expression were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot.. A decreased expression of COX-2 in human osteosarcoma cells significantly inhibited the growth and decreased the migration ability of SaOS2 cells. Furthermore, it also reduced VEGF, EGF and bFGF mRNA and protein expression.. The COX-2 signaling pathway may provide a novel therapeutic target for the treatment of human osteosarcoma.

Topics: Bone Neoplasms; Cell Line, Tumor; Cyclooxygenase 2; Epidermal Growth Factor; Fibroblast Growth Factor 2; Gene Knockdown Techniques; Humans; Osteosarcoma; RNA Interference; RNA, Small Interfering; Signal Transduction; Vascular Endothelial Growth Factor A

Osteosarcoma (OS) is a kind of malignancy wherein the tumor cells form malignant bone-like or bone tissue. Tenascin-C (TN-C), an important extracellular matrix (ECM) protein, plays an indispensable role in tumor development. However, its regulatory factors, expression, and function in OS pathological process have not been studied extensively. Expression of TN-C is induced by growth factors as well as mechanical strain in fibroblast. So we asked whether mechanical stain and growth factors could induce TN-C expression in OS as well as which pathways were involved in those processes. We found that when mechanical strain was applied to OS cells cultured on silicone membrane, TN-C mRNA and protein levels were increased 10-fold within 8 h compared to the resting control. Likewise, when epidermal growth factors (EGFs) and insulin-like growth factor (IGF-1) were added to cells, TN-C mRNA levels increased six fold and eightfold, respectively, within 24h compared to the control. Growth factors (EGF and IGF-1) and mechanical strain had additive effects on the induction of TN-C mRNA expression in OS. Both ROCK-I/II inhibitor and MEK-1 inhibitor inhibited TN-C induction by EGF or IGF-1, while only ROCK-I/II inhibitor had a strong subdued effect on TN-C induction by mechanical strain. Taken together, our findings suggest that growth factors and mechanical strain can induce TN-C in OS through different pathways additively.

Topics: Bone Neoplasms; Cell Line, Tumor; Epidermal Growth Factor; Fibroblasts; Gene Expression Regulation, Neoplastic; Humans; MAP Kinase Kinase 1; Mechanotransduction, Cellular; Osteosarcoma; Protein Kinase Inhibitors; rho-Associated Kinases; Somatomedins; Stress, Physiological; Tenascin; Up-Regulation

Ewing's sarcoma family of tumours (ESFT) is a malignant small round-cell tumour of the bone and soft tissues. It is characterised by a strong tendency to invade and form metastases. The microenvironment of the bone marrow is a large repository for many growth factors, including the basic fibroblast growth factor (bFGF). However, the role of bFGF in the invasive and metastatic phenotype of ESFT has not been investigated.. The motility and invasion of ESFT cells were assessed by a wound-healing assay, chemotaxis assay, and invasion assay. The expression and activation of FGF receptors (FGFRs) in ESFT cell lines and clinical samples were detected by RT-PCR, western blotting, and immunohistochemistry. The morphology of ESFT cells was investigated by phase-contrast microscopy and fluorescence staining for actin. Activation of Rac1 was analysed by a pull-down assay.. bFGF strongly induced the motility and invasion of ESFT cells. Furthermore, FGFR1 was found to be expressed and activated in clinical samples of ESFT. Basic FGF-induced cell motility was mediated through the FGFR1-phosphatidylinositol 3-kinase (PI3K)-Rac1 pathway. Conditioned medium from bone marrow stromal cells induced the motility of ESFT cells by activating bFGF/FGFR1 signalling.. The bFGF-FGFR1-PI3K-Rac1 pathway in the bone microenvironment may have a significant role in the invasion and metastasis of ESFT.

Topics: Bone Marrow; Cell Line, Tumor; Cell Movement; Cytoskeleton; Enzyme Activation; Epidermal Growth Factor; Fibroblast Growth Factor 2; Humans; Neoplasm Invasiveness; Neoplasm Metastasis; Osteosarcoma; Phosphatidylinositol 3-Kinases; Polymerase Chain Reaction; rac1 GTP-Binding Protein; Receptor, Fibroblast Growth Factor, Type 1; Receptor, Fibroblast Growth Factor, Type 3; Recombinant Proteins; Sarcoma, Ewing; Wound Healing

We recently developed a new tetracycline-inducible gene switch employing the tetracycline operator-containing hCMV major immediate-early promoter and the tetracycline repressor, tetR, rather than the previously used tetR-mammalian cell transcription factor fusion derivatives.. The present study demonstrates that this tetR-mediated transcription repression system can function as a powerful gene switch for On-and-Off regulation of therapeutic gene expression in ex vivo gene transfer protocols. Firstly, for achieving regulated gene expression in a localized tissue environment, R11/OEGF cells, a stable line that expresses hEGF under the control of the tetR-mediated transcription repression switch, were transplanted into porcine full-thickness wounds enclosed by wound chambers.. By topically applying tetracycline in wound chambers at various concentrations or at different time points post-transplantation, the levels and timing of hEGF expression in transplanted wounds could be reversibly regulated by tetracycline. Over 3000-fold induction in hEGF expression was achieved in the local wound microenvironment. Secondly, R11/OEGF cells were intramuscularly injected into NCr outbread nude mice to test the efficacy of intermittent systemic gene delivery of a soluble peptide(s).. Basal circulating hEGF was undetectable and induced up to at least 1,500-fold after administration of tetracycline. Furthermore, the timing and duration of hEGF expression could be finely adjusted by the presence or the absence of tetracycline in the drinking water.

Topics: Animals; Anti-Bacterial Agents; Cell Line, Tumor; Cytomegalovirus; Epidermal Growth Factor; Female; Gene Expression; Gene Transfer Techniques; Humans; Male; Mice; Mice, Nude; Osteosarcoma; Promoter Regions, Genetic; Skin; Sus scrofa; Tetracycline; Transgenes; Wounds and Injuries

The cell adhesion molecule T-cadherin is an unusual member of the cadherin superfamily that lacks a cytoplasmic domain, binding instead to the cell membrane via a glycophosphatidyl inositol anchor. T-cadherin is a receptor for hexameric Acrp30/adiponectin and binds low-density lipoproteins in endothelial cells. T-cadherin is expressed widely in the brain and cardiovascular system, but expression is absent or decreased in several cancers. Little is known about the mechanisms and factors that control T-cadherin expression. Therefore, to investigate regulation of T-cadherin expression, we analysed 3.9 kb of the 5'-flanking region of human T-cadherin for promoter activity and identified potential transcription factor binding sites. Western blotting and a quantitative real-time RT-PCR assay developed for T-cadherin showed that estradiol, progesterone, EGF, dexamethasone and factors in serum were involved in transcriptional and post-transcriptional regulation of T-cadherin in human osteosarcoma cells; the effects observed were opposite to those described for T-cadherin's ligand, adiponectin. The data suggest that T-cadherin is regulated in a complex manner indicative of a role in hormone and drug-induced changes in bone morphology and pathology.

Topics: 5' Flanking Region; Animals; Cadherins; Cell Adhesion; Cell Line, Transformed; Cell Line, Tumor; Cell Transformation, Viral; Chlorocebus aethiops; COS Cells; Dexamethasone; Epidermal Growth Factor; Estradiol; Gene Expression Regulation; Genes, Reporter; Glucocorticoids; Hormones; Humans; Luciferases; Osteosarcoma; Progesterone; Promoter Regions, Genetic; RNA, Messenger

Thrombospondin-1 (TSP-1), a multifunctional matrix protein, affects tumor growth through modulation of angiogenesis and other stromal biological functions. In several of nine human cancer cell lines derived from liver, brain, pancreas, and bone, expression of TSP-1 was up-regulated in response to the two most representative growth factors, epidermal growth factor (EGF) and transforming growth factor beta1 (TGFbeta1). Expression of TSP-1 was markedly enhanced in hepatic HuH-7 cells by EGF but not by TGFbeta1. In contrast, expression of TSP-1 was markedly enhanced by TGFbeta1, but not by EGF, in osteosarcoma MG63 cells. EGF induced activation of TSP-1 promoter-driven luciferase activity in HuH-7 cells, and the elements between -267 and -71 on the 5' region of TSP-1 gene containing two GC boxes to which Sp1 bound, were found to be responsible for the promoter activation by EGF. However, EGF did not alter TSP-1 mRNA stability in hepatic cells. On the other hand, no such enhancement of the TSP-1 promoter activity by TGFbeta1 appeared in MG63 cells. Enhanced expression of TSP-1 by TGFbeta1 in MG63 cells was partially blocked by exogenous addition of SB203580, an inhibitor of p38 mitogen-activated protein kinase. TGFbeta was found to induce marked elongation of TSP-1 mRNA longevity in osteosarcoma cells when mRNA degradation was assayed in the presence of alpha-amanitin. The up-regulation of TSP-1 by EGF and TGFbeta might play a critical role in modulation of angiogenesis and formation of matrices in tumor stroma.

Topics: Bone Neoplasms; Carcinoma, Hepatocellular; Epidermal Growth Factor; Gene Expression Regulation; Humans; Liver Neoplasms; Osteosarcoma; RNA Stability; Thrombospondin 1; Transcriptional Activation; Transforming Growth Factor beta; Tumor Cells, Cultured; Up-Regulation

With the aim of identifying innovative therapeutic strategies for osteosarcoma patients who are refractory to conventional chemotherapy, we analyzed the in vitro effects of the blockage of autocrine circuits. Since the insulin-like growth factor-I receptor (IGF-IR)-mediated loop is relevant to the growth of osteosarcoma, we analyzed the activity of the IGF-IR-blocking antibody alphaIR3 in both sensitive and multidrug-resistant osteosarcoma cell lines. Only limited effects, however, were observed, suggesting the simultaneous existence of other autocrine circuits. Indeed, in a representative panel of 12 human osteosarcoma cell lines, in addition to the IGF-IR-mediated circuit, we demonstrated also a loop mediated by epidermal growth factor receptor as well as the presence of nerve growth factor, low-affinity nerve growth factor receptor as well as tyrosine receptor kinase A in the great majority of osteosarcomas. Therapies based on the inhibition of single circuits may have only limited effects in osteosarcoma, whereas the use of suramin, a drug which, besides other activities, non-selectively interferes with the binding of growth factors to their receptors, appears as a promising alternative, in both sensitive and drug-resistant osteosarcoma cells.

Topics: Antineoplastic Agents; Autocrine Communication; Becaplermin; Bone Neoplasms; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Epidermal Growth Factor; Humans; Insulin-Like Growth Factor I; Insulin-Like Growth Factor II; Neoplasm Proteins; Nerve Growth Factors; Osteosarcoma; Platelet-Derived Growth Factor; Proto-Oncogene Proteins c-sis; Receptor, IGF Type 1; Receptors, Growth Factor; Suramin; Transforming Growth Factor alpha; Tumor Cells, Cultured

A model using chemically permeabilized cells was developed to examine mechanisms that regulate protein tyrosine phosphorylation in osteoblastic cells. Using either permeabilized UMR106 osteoblastic or A431 (reference) cells, epidermal growth factor (EGF)-induced cellular tyrosine phosphorylation, and whether there are previously unrecognized interactions between this transduction pathway and Ca2+- or G protein-dependent signalling pathways, were investigated. Both permeabilized cell types, when maintained in non-supplemented cytoplasmic substitution solution (basic CSS), responded to EGF (1-100 ng/ml) with dose-dependent increases in tyrosine phosphorylation. A complex and time-dependent pattern of phosphotyrosine-containing proteins resulted, but the profile of tyrosine phosphorylated proteins was appreciably less complex than in intact cells. Supplementation of basic CSS with MgATP restored the normal complexity of the profiles for EGF-induced tyrosine phosphorylation proteins in both permeabilized cell lines and produced a more sustained accumulation of phosphoprotein products in A431 cells. Adding Ca2+ (< or = 10(-6) M), with or without exogenous MgATP, dose-dependently attenuated EGF-induced tyrosine phosphorylation of EGF receptors (EGFR) and other substrates in UMR106 cells, but was less effective in A431 cells. In both cell types, genistein, an inhibitor of tyrosine kinases, was more effective in attenuating EGF-induced receptor tyrosine phosphorylation in permeabilized cells. Similarly, orthovanadate, an inhibitor of protein tyrosine phosphatases, stimulated the accumulation of phosphoprotein products more effectively in permeabilized cells. Thus, the permeabilization preserves many features of intact cells while facilitating manipulation of intracellular conditions. NaF reproducibly produced a significant vanadate-like action in permeabilized cells that was somewhat stronger than its effect on intact cells. In contrast, the well-known inhibition of tyrosine phosphorylation by phorbol 12-myristate 13-acetate (PMA) was less effective in permeabilized cells than in intact cells; these actions of PMA were Ca2+-dependent. In addition, guanylyl-imidodiphosphate (Gpp(NH)p) attenuated tyrosine phosphorylation in UMR106 cells, and this effect was specifically blocked by guanosine 5'-O-(2-thiodiphosphate) (GDPbetas). These results strongly suggest that there is crosstalk between EGFR-activated tyrosine phosphorylation/dephosphorylation pathways and both C

Topics: Adenosine Triphosphate; Animals; Calcium; Calcium Signaling; Carcinoma; Cell Membrane Permeability; Culture Media; Dose-Response Relationship, Drug; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Genistein; GTP-Binding Proteins; Humans; Osteoblasts; Osteosarcoma; Phosphoproteins; Phosphorylation; Protein Tyrosine Phosphatases; Protein-Tyrosine Kinases; Rats; Signal Transduction; Time Factors; Tumor Cells, Cultured; Tyrosine; Vanadates

This article describes the first (to our knowledge) tetracycline-inducible regulatory system that demonstrates that the tetracycline repressor (tetR) alone, rather than tetR-mammalian cell transcription factor fusion derivatives, can function as a potent trans-modulator to regulate gene expression in mammalian cells. With proper positioning of tetracycline operators downstream of the TATA element and of human epidermal growth factor (hEGF) as a reporter, we show that gene expression from the tetracycline operator-bearing hCMV major immediate-early enhancer-promoter (pcmvtetO) can be regulated by tetR over three orders of magnitude in response to tetracycline when (1) the reporter was cotransfected with tetR-expressing plasmid in transient expression assays, and (2) the reporter unit was stably integrated into the chromosome of a tetR-expressing cell line. This level of tetR-mediated inducible gene regulation is significantly higher than that of other repression-based mammalian cell transcription switch systems. In an in vivo porcine wound model, close to 60-fold tetR-mediated regulatory effects were detected and it was reversed when tetracycline was administered. Collectively, this study provides a direct implementation of this tetracycline-inducible regulatory switch for controlling gene expression in vitro, in vivo, and in gene therapy.

Topics: Animals; Base Sequence; Chlorocebus aethiops; Cytomegalovirus; Epidermal Growth Factor; Female; Gene Expression Regulation; Gene Transfer Techniques; Genes, Immediate-Early; Genes, Reporter; HeLa Cells; Herpes Simplex Virus Protein Vmw65; Humans; Molecular Sequence Data; Operator Regions, Genetic; Osteosarcoma; Recombinant Fusion Proteins; Repressor Proteins; Swine; TATA Box; Tetracycline; Transcription, Genetic; Tumor Cells, Cultured; Vero Cells; Wounds and Injuries

We investigated the characteristics of nucleolar organizer regions (NORs) and epidermal growth factor receptor (EGFR) on differentiated Dunn osteosarcoma in response to dibutyryl cyclic adenosine 3',5'-monophosphate (Bt2cAMP). In the presence of 3 mM Bt2cAMP, the mean number of NORs was significantly decreased in 24 hrs and 48 hrs compared with the untreated group. Also, EGFR affinity was decreased and the EGFR number was increased in response to the cellular differentiation by Bt2cAMP. The decrease in EGFR affinity might be considered as an indicator of differentiation or the mature state of the cells. Thus, the present study provides a new clue to support differentiation of osteosarcoma cells from the viewpoint of NORs findings and EGFR analysis as a differentiation marker.

Topics: Animals; Biomarkers, Tumor; Bone Neoplasms; Bucladesine; Cell Differentiation; DNA, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Female; Iodine Radioisotopes; Mice; Mice, Inbred C3H; Nucleolus Organizer Region; Osteosarcoma; Silver Staining; Tumor Cells, Cultured

This study was designed to test the hypothesis that treatment of human bone cells with mitogenic concentrations of fluoride would lead to an increase in the steady state level of tyrosyl phosphorylation of specific cellular proteins. With an immunoblot assay method, it was found that mitogenic concentrations of fluoride (i.e. 50-200 mumol/L) induced a dose- and time-dependent increase in the level of tyrosyl phosphorylation of at least 13 cellular proteins in both normal human bone cells and human TE85 osteosarcoma cells. Time-course studies revealed that a statistically significant increase in tyrosyl phosphorylation of these 13 cellular proteins in human bone cells was observed after 3-6 h of fluoride treatment and was sustained for up to 24 h. This time course was not compatible with a direct activation of tyrosyl kinases, as epidermal growth factor, which activates tyrosyl kinase activity, induced an immediate and acute response that was rapidly reversible within 1 h. Although fluoride increased the steady state tyrosyl phosphorylation of the cellular proteins in human bone cells, the same micromolar doses of fluoride had no effect on human skin fibroblasts, which are fluoride-nonresponsive cells. The effects of fluoride were rapidly reversible in the absence of fluoride and could be acutely potentiated by pretreatment with epidermal growth factor. In summary, we have shown for the first time that mitogenic concentrations (i.e. 50-200 mumol/L) of fluoride increased the steady state level of tyrosyl phosphorylation of at least 13 cellular proteins in human bone cells, and that the increases were relatively show in onset and sustained. In conclusion, these findings are consistent with the hypothesis that the osteogenic actions of fluoride are mediated at least in part by an inhibition of the activity of one or more fluoride-sensitive phosphotyrosyl protein phosphatases in human bone cells.

Topics: Bone and Bones; Dose-Response Relationship, Drug; Drug Synergism; Epidermal Growth Factor; Fluorides; Humans; Immunoblotting; Kinetics; Molecular Weight; Osteosarcoma; Phosphoproteins; Phosphorylation; Phosphotyrosine; Tumor Cells, Cultured

The gene encoding PTH-related peptide (PTHrP), a protein that plays a primary role in the development of humoral hypercalcemia of malignancy, is widely expressed in normal and neoplastic tissues. This study demonstrates that expression of the PTHrP gene has features of early response genes, including up-regulation after serum repletion of serum-starved ROS 17/2.8 (rat osteosarcoma) cells. The PTHrP messenger RNA (mRNA) levels were induced within 30 min and peaked at 4 h. Elevated mRNA levels were accompanied by an increase in secreted PTHrP. The serum effects on PTHrP mRNA levels were blocked by actinomycin D, suggesting a requirement for gene transcription. Nuclear run-on assays revealed a 3-fold increase in PTHrP gene transcription 4 h after exposure to serum. Deletions of the 5' flanking sequence of the rat PTHrP gene fused to the chloramphenicol acetyltransferase gene and transfected into ROS 17/2.8 cells showed that the serum-responsive region is located between -1.05 kb and -0.3 kb upstream of the transcription start site. PTHrP mRNA levels were also induced by cycloheximide, another feature common to early response genes. The PTHrP mRNA half-life in serum-starved cells was 56 min. Serum treatment prolonged the half-life 2.7-fold, suggesting serum-induced stabilization of the mRNA. Insulin and epidermal growth factor also induced PTHrP mRNA expression in a time-dependent manner analogous to serum, indicating that the effects of serum may be mediated, at least partially, through these agents. In summary, serum up-regulated PTHrP mRNA expression through both transcriptional and posttranscriptional mechanisms. This rapid stimulation by growth factors suggests that PTHrP may contribute to the early cellular response after growth factor stimulation.

Topics: Animals; Blood Physiological Phenomena; Cycloheximide; Epidermal Growth Factor; Gene Expression; Insulin; Neoplasm Proteins; Osteosarcoma; Parathyroid Hormone-Related Protein; Protein Processing, Post-Translational; Proteins; Rats; RNA, Messenger; Transcription, Genetic; Tumor Cells, Cultured

Topics: Analysis of Variance; Animals; Calcium Hydroxide; Dose-Response Relationship, Drug; Drug Synergism; Epidermal Growth Factor; Osteoblasts; Osteosarcoma; Rats; Spectrometry, Fluorescence; Tumor Cells, Cultured

Interleukin-1 (IL-1) is a potent bone resorbing cytokine with diverse biological effects. We previously reported that IL-1 inhibits PDGF-AA-induced biological activities including PDGF-AA-induced tyrosyl phosphorylation. In the present studies, we first investigated and compared the tyrosyl phosphorylation pattern induced by EGF, IGF-1, PDGF-AA, and bFGF in human osteoblastic cells. We then examined the effect of IL-1 on the tyrosyl phosphoproteins induced by each ligand. Immunoblot analyses show that EGF, IGF-1, and PDGF-AA each elicit a different pattern of tyrosyl phosphorylated proteins in normal human osteoblastic cells. IL-1 beta inhibits PDGF-AA induced autophosphorylation by down-regulation of the PDGF-alpha receptor, as demonstrated by immunoprecipitation experiments. For other ligand-induced tyrosyl phosphoproteins, IL-1 beta reduced the intensity of EGF-induced pp55,000, and IGF-1 induced pp185,000 and pp175,000. These experiments indicate that IL-1 inhibits phosphorylation of specific proteins induced by growth factors. By using inhibitors of secondary message pathways, we determined that the inhibitory effect of IL-1 beta on PDGF-AA receptor binding and receptor tyrosyl autophosphorylation was not dependent on protein kinase A, protein kinase C, or the formation of prostaglandins. These data suggest the existence of an alternative pathway that may participate in IL-1 beta signaling.

Topics: Bone Neoplasms; Bone Resorption; Cell Division; Electrophoresis, Polyacrylamide Gel; Epidermal Growth Factor; Fibroblast Growth Factor 2; Fibroblasts; Humans; Interleukin-1; Osteoblasts; Osteosarcoma; Phosphorylation; Platelet-Derived Growth Factor; Proteins; Receptors, Growth Factor; Receptors, Platelet-Derived Growth Factor; Signal Transduction; Tumor Cells, Cultured; Tyrosine

Epidermal growth factor (EGF) induces a rapid increase in the phosphorylation of extracellular signal-regulated kinases (ERKs) in the human osteosarcoma osteoblastic cell line G292 and in primary cultures of rat osteoblastic cells. This phosphorylation is transient and time-dependent. Maximal stimulation is attained within 1 min in G292 and within 5 min in rat osteoblastic cells. Enzymatic activity in G292 cells is also induced rapidly after EGF stimulation. Western blot analysis revealed that enhancement of the phosphorylation of ERKs in the EGF-stimulated cells is not due to an increase in ERK protein, since EGF-treatment does not lead to an increase in the absolute amount of ERKs present even after 2 days of stimulation. The pattern of expression of the ERKs observed in the two cell types differs in the apparent molecular weights observed. The most slowly migrating immunoreactive protein (approximately 45 kDa) in normal rat osteoblastic cells is ERK1, identified by an ERK1-selective antiserum. The same antiserum reacts only weakly with one of the ERK proteins (44 kDa) blotted from the human osteosarcoma cell line G292. Phorbol 12-myristate 13-acetate (PMA) is also capable of inducing ERK phosphorylation, albeit to a lasser degree. The combination of PMA and EGF does not produce a greater response than EGF alone. The role of protein kinase C (PKC) in the EGF-stimulated ERK signaling pathway was further examined by inhibition of PKC with the staurosporine analog, CGP41251, and by down-regulation of PKC via chronic treatment with PMA. Chronic PMA treatment results in a partial inhibition of the EGF-mediated phosphorylation. CGP41251 completely abolishes the increased ERK activity produced by PMA, but the effect of EGF in this regard is potentiated. We conclude that PKC and EGF act through parallel pathways to stimulate ERK phosphorylation and activity. The inhibitor studies, in addition, indicate that activation of PKC may moderate the actions of the EGF pathway via a tonic inhibitory feedback.

Topics: Animals; Calcium-Calmodulin-Dependent Protein Kinases; Enzyme Activation; Epidermal Growth Factor; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Osteoblasts; Osteosarcoma; Phosphorylation; Protein Kinase C; Rats; Signal Transduction; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

The expression of both epidermal growth factor (EGF) and platelet-derived growth factor (PDGF), and of their receptors (EGFR and PDGFR) was immunohistochemically examined in 37 cases of osteosarcoma. Furthermore, immunostaining for p53 protein and Ki-67 antigen by MIB-1 was carried out and compared with the above results. EGFR (81%) expressed more often than EGF (51%) and the expression of EGF and EGFR, and PDGF and PDGFR were recognized in 49% and 38%, respectively. In eleven cases (30%), the expression of both growth factors and their receptors was combined. Anaplastic osteosarcoma showed higher MIB-1 index than osteoblastic and fibroblastic subtypes (P < 0.05). High grade osteosarcomas (G3 and G4) revealed higher MIB-1 index compared with low grade tumors (G1 and G2). PDGF positive tumors (MIB-1 index: 20.0) showed significantly higher proliferation compared with PDGF negative tumors (MIB-1 index: 6.5) (P < 0.01). Five out of 37 cases (13.5%) showed positive immunoreaction for p53. There was no correlation of p53 status with MIB-1 index and the expression of growth factors or their receptors. Our results suggest that PDGF expression may be an important mediator of cell proliferation control, via an autocrine mechanism, in human osteosarcoma.

Topics: Antibodies, Monoclonal; Bone Neoplasms; Cell Division; Epidermal Growth Factor; ErbB Receptors; Growth Substances; Humans; Immunohistochemistry; Osteosarcoma; Platelet-Derived Growth Factor; Receptors, Platelet-Derived Growth Factor; Tumor Suppressor Protein p53

Sequence information for aspartyl beta-hydroxylase (AspH), which specifically hydroxylates one Asp or Asn residue in certain epidermal growth factor (EGF)-like domains of a number of proteins, is so far only described for bovine species. We have isolated a 4.3-kb cDNA encoding the human AspH (hAspH) by immunoscreening of a human osteosarcoma (MG63) cDNA library in lambda ZAP with an antiserum raised against membrane fractions of these cells. Northern blot analyses revealed two transcripts with lengths of 2.6 and 4.3 kb. The deduced amino acid (aa) sequence of this cDNA encodes a protein of 757 aa (85 kDa). Comparison with the deduced bovine AspH (bAspH) aa sequence showed striking differences in the N-terminal portion of this protein. In vitro transcription and translation in the presence of canine pancreas microsomes yielded a 56-kDa protein. Western blot analyses of membrane fractions from MG63 cells with AspH-specific antibodies revealed a protein of the same M(r). These results suggest a posttranslational cleavage of the catalytic C terminus in the lumen of the endoplasmic reticulum.

Topics: Amino Acid Sequence; Animals; Base Sequence; Blotting, Northern; Bone Neoplasms; Cattle; Cell Membrane; Cloning, Molecular; Epidermal Growth Factor; Hominidae; Humans; Immune Sera; Mixed Function Oxygenases; Molecular Sequence Data; Osteosarcoma; Protein Biosynthesis; Sequence Homology, Amino Acid; Transcription, Genetic; Tumor Cells, Cultured

Local factors play an important role in the regulation of bone metabolism. The homologous and heterologous desensitization of responses to these factors may be crucial in the modulation of bone cell signaling. In this study, the effects and interactions of endothelin-1 (25 nM), alpha-thrombin (0.9 microM), epidermal growth factor (40 nM), prostaglandin E1 (5 microM), and prostaglandin F1 alpha (5 microM) were examined on calcium signaling in UMR-106 rat osteoblastic osteosarcoma cells. Intracellular calcium was measured using fluo-3 fluorescent dye. All agents elicited calcium transients at these concentrations and showed homologous desensitization to their repeated administration. Preincubation for 60 minutes with 500 microM monodansylcadaverine and 30 minutes or 24 h preincubation with 0.5 microM indomethacin did not affect homologous desensitization, suggesting that neither the internalization of receptors nor prostaglandins are involved in this event. Pretreatment for 3 minutes with 2 microM 4 beta-phorbol-12 beta, 13 alpha-dibutyrate significantly reduced the calcium elevations elicited by the first application of these compounds, whereas an inactive phorbol, 12,13-didecanoate, had no effect. Pretreatment for 4 minutes with 0.5 microM forskolin decreased the calcium signal response to PGE1 only. Pretreatment with endothelin-1 for 3 minutes significantly decreased the calcium signals elicited by epidermal growth factor and alpha-thrombin. Prior administration of endothelin-1 significantly increased prostaglandin E1-stimulated calcium transients, whereas prostaglandin F1 alpha responses were not affected. Preincubation with indomethacin did not alter any of the interactions. Responses to endothelin-1 were not significantly altered by 2-3 minutes pretreatment with the other factors, nor was there cross-desensitization among the other factors. The results could indicate that endothelin-1 has a unique and specific role in the modulation of bone cell signaling.

Topics: Alprostadil; Animals; Cadaverine; Calcium; Endothelins; Epidermal Growth Factor; Indomethacin; Osteoblasts; Osteosarcoma; Phorbol Esters; Prostaglandins F; Rats; Signal Transduction; Thrombin; Tumor Cells, Cultured

This study sought to understand the role of epidermal growth factor receptor (EGF-R) in periodontal ligament (PDL) fibroblasts. Rat PDL fibroblastic cells and ROS 17/2.8 cells (highly differentiated osteoblastic osteosarcoma cells) were cultured and treated with transforming growth factor-alpha (TGF-alpha), EGF, dexamethasone (Dex) or a combination of EGF and Dex. Alkaline phosphatase (ALP) activity, an early differentiation marker for mineralized tissue-forming cells, was measured using p-nitrophenylphosphate as a substrate. For Scatchard analysis of [125I]-EGF binding, cells were incubated in Dulbecco's modified Eagle's medium containing 0.2% bovine serum albumin and 0-64 ng/ml of [125I]-EGF for 4 h at 4 degrees C. Also, the synthesis of EGF-R protein and the expression of mRNA for EGF-R were measured by immunoprecipitation and Northern blot analysis, respectively. Untreated PDL fibroblastic cells showed a gradual increase in spontaneous ALP activity from 32.4 U/10(6) cells at 2 days to 49.6 U/10(6) cells at 7 days of culture. ALP activity was further increased to 70.8 U/10(6) cells at 7 days after treatment with Dex, whereas EGF treatment reduced it to 19.4 U/10(6) cells. Culture of PDL fibroblastic cells in the presence of a combination of Dex and EGF decreased the Dex-induced ALP activity from 70.8 U to 41.8 U/10(6) cells at 7 days. A similar inhibitory effect on ALP activity was found after treatment with TGF-alpha. In contrast, ROS cells maintained a high ALP activity (1748 U/10(6) cells) throughout culture, unaffected by EGF. Scatchard analysis demonstrated that PDL fibroblastic cells have both high- and low-affinity forms of EGF-R, while ROS cells did not have any detectable EGF-R. Treatment of PDL cells with Dex for 2 days decreased the synthesis of EGF-R protein, the expression of EGF-R mRNA and the number of EGF-R. In contrast, EGF treatment increased the expression of EGF-R mRNA. These data suggest that PDL fibroblastic cells express numerous EGF-R, but the number decreases during their differentiation into mineralized tissue-forming cells under the influence of Dex. Thus, EGF-R may function in the stabilization of phenotype in PDL fibroblastic cells.

Topics: Alkaline Phosphatase; Animals; Autoradiography; Cell Differentiation; Cells, Cultured; Dexamethasone; Endocytosis; Epidermal Growth Factor; ErbB Receptors; Female; Fibroblasts; Gene Expression; Osteosarcoma; Periodontal Ligament; Phenotype; Rats; Rats, Sprague-Dawley; Tumor Cells, Cultured; Up-Regulation

Prostaglandin E2 (PGE2), PTH, and epidermal growth factor (EGF) are potent regulators of osteoblast proliferation. In UMR 106-01 rat osteosarcoma cells with osteoblast-like features, PGE2 and PTH inhibit, while EGF stimulates, mitogenesis. Both PGE2 and PTH increase intracellular cAMP levels, cytosolic calcium, and inositol phosphate turnover. In a variety of cell types, EGF mediates its effects in part via activation of receptor protein-tyrosine kinase and other protein kinases, such as protein kinase-C. The nuclear mechanisms of PGE2, PTH, and EGF regulation of osteoblast proliferation are unknown. Accordingly, we have examined the effects of these agents on mitogenesis, second messenger generation, and primary response genes, which may link second messenger activation to subsequent alterations in gene expression. Northern blot analysis of mRNA from UMR 106-01 cells treated for 3 h with 2 microM PGE2, 10 nM PTH, or 10 ng/ml EGF in the presence of cycloheximide demonstrated that all three agents induced the expression of c-fos and c-jun mRNA. In contrast, only EGF stimulated cellular proliferation and induced Egr-1 mRNA. Also, unlike PGE2 and PTH, EGF did not increase intracellular cAMP levels. c-fos mRNA was induced by treatment with 50 ng/ml tetradecanoyl phorbol acetate or by 40 ng/ml forskolin, while induction of Egr-1 mRNA was stimulated by treatment with tetradecanoyl phorbol acetate, but not forskolin. Thus, EGF signal transduction differs from that of PGE2 and PTH in UMR 106-01 osteoblast-like cells, in that EGF does not stimulate the protein kinase-A second messenger system, but causes activation of Egr-1, a primary response gene that may play a role in the mitogenic effect of EGF.

Topics: Animals; Bone Neoplasms; Calcium; Colforsin; Cyclic AMP; Cycloheximide; Dinoprostone; DNA-Binding Proteins; Early Growth Response Protein 1; Enzyme Activation; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Genes, fos; Genes, jun; Immediate-Early Proteins; Inositol Phosphates; Mitosis; Osteoblasts; Osteosarcoma; Parathyroid Hormone; Protein Kinases; Radioimmunoassay; Rats; RNA, Messenger; Signal Transduction; Tetradecanoylphorbol Acetate; Thymidine; Transcription Factors; Tritium; Tumor Cells, Cultured; Zinc Fingers

The human osteosarcoma cell line (OST-1-PF) can grow in protein-free Coon's modified Ham's F12 medium. Growth of the cells in protein-free medium was partially density-dependent and partially depressed by medium change. An extract and conditioned medium of OST-1-PF cells contained high mitogenic activity for BALB/c3T3 cells. The growth factor in the cells was purified and identified as a basic fibroblast growth factor (bFGF)--like factor on the basis of its elution profile on heparin-affinity chromatography and the result of immunoblotting. An unidentified factor in a conditioned medium eliciting most of the DNA synthesis-stimulating activity showed a weak affinity for heparin. Various additions, including serum and growth factors, stimulated the growth of OST-1-PF cells in protein-free medium. Of these factors, epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), acidic fibroblast growth factor (aFGF) and bFGF were the most potent mitogens. High-affinity receptors of EGF and FGF were found on the surface of these cells. These results indicate that autonomous growth of OST-1-PF cells in protein-free medium is mainly controlled by an intracellular mechanism.

Topics: Cell Division; Culture Media, Serum-Free; Epidermal Growth Factor; Fibroblast Growth Factors; Humans; Osteosarcoma; Transforming Growth Factor alpha; Tumor Cells, Cultured

The regulation of collagenase gene expression in the human osteosarcoma-derived osteoblastic cell lines MG-63, U2-OS and human fibroblast cell line IMR-90 was investigated by Northern blot analysis. Exposure of quiescent MG-63, U2-OS and IMR-90 cells to 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and 10% fetal calf serum (FCS) resulted in the induction of mRNA encoding collagenase. Epidermal growth factor (EGF) induced collagenase mRNA in the IMR-90 cell but not in the MG-63 and U2-OS cells. In the IMR-90 and MG-63 cells, EGF stimulated the transcription of the c-fos and c-jun genes encoding the transcriptional factors which interact directly with the promoter region of the human collagenase gene. Parathyroid hormone and 1,25-dihydroxy-vitamin D3 did not increase the collagenase mRNA level in both osteosarcoma cells. Recombinant interleukin-1 beta (rIL-1 beta) induced collagenase and c-jun but not c-fos mRNA in the MG-63 cell. The induction by rIL-1 beta was blocked by cycloheximide and dexamethasone. Transforming growth factor beta 1 blocked the FCS-induced collagenase gene expression but partially inhibited the rIL-1 beta-induced gene expression in the MG-63 cell. These results suggest that the collagenase activity is regulated not only by post-translational modification but also at the transcriptional level by the various factors in bone.

Topics: Epidermal Growth Factor; Fibroblasts; Gene Expression Regulation, Enzymologic; Genetic Code; Humans; Interleukin-1; Microbial Collagenase; Osteoblasts; Osteosarcoma; Recombinant Proteins; RNA, Messenger; Transcription, Genetic; Tumor Cells, Cultured

In attempt to study the mechanism of F(-)-induced, osteoblast-mediated bone formation, we tried to show the characteristics of Al-F complex-induced mitogenesis in osteoblastic cells. The MOB 3-4-F2 cell line, an osteoblast-like cell line derived from neonatal mouse calvaria, responded to F- (1-2 mM) combined with Al3+ and epidermal growth factor (EGF, 0.01-100 ng/ml) with increased DNA synthesis. Of the several types of Al-F complexes, AlF4- is thought to act as a mitogenic factor. On the other hand, NaF at high concentrations (greater than 2 mM) markedly decreased cell viability. The AlF(4-)-stimulated DNA synthesis at least with a delay of 48 hr, while EGF stimulated DNA synthesis within a few hours (4-6 hr). Both 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7) and staurosporine, inhibitors of protein kinase C (PKC), further enhanced DNA synthesis in AlF(4-)-treated cells, whereas 12-O-tetradecanoyl-13-acetate (TPA), an activator of PKC, decreased the DNA synthesis. In EGF-treated cells, staurosporine and TPA, but not H-7, decreased DNA synthesis. In addition, indomethacin, an inhibitor of cyclooxygenase, partly inhibited the EGF-induced mitogenesis, which, however, was restored by addition of PGE2. AlF4-, as well as EGF, stimulated the release of arachidonic acid and its metabolites. Indomethacin failed to inhibit the AlF(4-)-induced mitogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Aluminum; Aluminum Compounds; Animals; Arachidonic Acid; Cell Line; DNA; Epidermal Growth Factor; Fluorides; Indomethacin; Mice; Osteoblasts; Osteosarcoma; Protein Kinase C; Tumor Cells, Cultured

Confluent cultures of rat bone cells synthesize several forms of secreted phosphoprotein 1 (SPP-1, osteopontin), the major phosphorylated forms of which migrate at 55 and 44 kDa on 15% cross-linked SDS-PAGE gels and correspond to the transformation-associated proteins pp 69 and pp 62. A clonal rat calvarial cell line (RCA 11), which expressed the highest level of SPP-1, produced only the 55 kDa form of the phosphorylated protein, whereas normal rat calvarial cells enriched in osteoblastic cells (RC IV cells) produced mostly the 55 kDa form, with small amounts of the 44 kDa form. In contrast, a 44 kDa form was the major [32PO4]-labelled SPP-1 synthesized by a rat osteocarcoma cell line (ROS 17/2.8 cells) with lesser amounts of the 55 kDa SPP-1. When [35S]methionine was used to measure protein synthesis, only the 55 kDa SPP-1 could be clearly detected in confluent cultures of each cell population, indicating that the 55 kDa SPP-1 is the prominent form of SPP-1 synthesized by each cell population. Following treatment of the normal rat bone cells for 24 h with osteotropic hormones (vit D3, PTH and RA), growth factors (PDGF, EGF, TGF-beta), a tumor promoter (TPA) and a plant lectin (Con A), the 55 kDa [35S]methionine labelled SPP-1 was increased 1.7-8.3-fold. Similar, but generally lower responses were observed in the clonal RCA 11 cell line, whereas the ROS 17/2.8 cells were more refractory, showing only a strong response to vit D3. In general, vit D3 produced the strongest stimulation in all populations with TGF-beta producing a good response in the non-transformed cells and RA in the RC IV cells. In contrast, PTH was inhibitory in both RCA 11 and ROS 17/2.8 cells. In most, but not all, cases the alteration in SPP-1 synthesis reflected similar changes in SPP-1 mRNA and in the intensity of the [32PO4]-labelled 55 kDa SPP-1. Collectively, these studies demonstrate that bone cells produce several forms of SPP-1 which are differentially regulated in normal and transformed cells through both transcriptional and posttranscriptional mechanisms.

Topics: Animals; Calcitriol; Cell Line; Cell Line, Transformed; Cells, Cultured; Concanavalin A; Epidermal Growth Factor; Growth Substances; Nucleic Acid Hybridization; Osteopontin; Osteosarcoma; Parathyroid Hormone; Platelet-Derived Growth Factor; Precipitin Tests; RNA, Messenger; Sialoglycoproteins; Tetradecanoylphorbol Acetate; Transcription, Genetic; Transforming Growth Factor beta; Tretinoin

The interaction of epidermal growth factor (EGF) and PTH with UMR 106 osteosarcoma cells was examined biochemically and morphologically. EGF inhibited PTH-stimulated adenylate cyclase activity in association with a reduction in PTH receptors and a decrease in the activity of the stimulatory guanyl nucleotide-binding protein (Gs). Universal inhibition of agonist-stimulated adenylate cyclase activity did not occur, inasmuch as EGF did not reduce prostaglandin E2-enhanced enzyme activity. The influence of EGF on PTH action correlated with its effect in the UMR 106 cell population of promoting entry into the cell cycle. Morphological analysis with radioautography indicated that both EGF and PTH receptors could be colocalized to certain UMR 106 cells, but that each were more abundantly distributed over discrete UMR 106 cell types. Based on the distribution of [3H]thymidine incorporation, EGF receptors were predominantly found over rapidly proliferating cells, whereas PTH receptors were most densely distributed over more quiescent cells. The results indicate that EGF and PTH receptors are localized over specific types within the heterogeneous population of UMR 106 cells and suggest that EGF may reduce PTH action in these cultures by enhancing the proliferation of progenitor cells lacking PTH receptors and reducing differentiation in this cell population, which leads to PTH receptor-enriched target cells. EGF and PTH receptors may, therefore, be useful as probes to examine both functional interactions and differentiation pathways among cells in osteoblast models in vitro and perhaps in vivo.

Topics: Adenylyl Cyclase Inhibitors; Animals; Cell Count; Dinoprostone; Epidermal Growth Factor; Osmolar Concentration; Osteosarcoma; Parathyroid Hormone; Peptide Fragments; Rats; Teriparatide; Thymidine; Time Factors; Tumor Cells, Cultured

We studied the binding and degradation of 125I-labeled epidermal growth factor (EGF) by UMR-106 osteosarcoma cells and the regulation of EGF receptor affinity for EGF by the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and by treatments that raise intracellular levels of cyclic AMP. Cell surface binding of [125I]EGF to A431 cells reached a plateau after a 30 minute incubation at 37 degrees C but was undetectable in UMR-106 cells. Degradation of [125I]EGF proceeded at a 50-fold higher rate in A431 cells on a per cell basis, but receptor-bound [125I]EGF was internalized and degraded at a 3.5-fold higher rate by UMR-106 cells on a per receptor basis. At 4 degrees C, [125I]EGF labeled a single class of surface binding sites in the UMR-106 cell. Treatment with TPA at 37 degrees C reduced subsequent cell surface binding of [125I]EGF at 4 degrees C a maximum of 80% with an IC50 of 1.25 ng/ml. Maximal TPA reduction of [125I]EGF binding was observed within 5-15 minutes and was due to a reduction in the affinity of cell surface receptors of [125I]EGF without a change in receptor density. Pretreatment of the cells for 4 h with 30 microM forskolin, 1 mM isobutylmethylxanthine (IBMX) plus 30 microM forskolin, or 1 mM IBMX plus 100 ng/ml parathyroid hormone (PTH) attenuated the loss in [125I]EGF binding caused by a subsequent dose of 10 ng/ml of TPA by 17% (p less than 0.0005), 39% (p less than 0.0002), and 35% (p less than 0.002), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 1-Methyl-3-isobutylxanthine; Colforsin; Cyclic AMP; Epidermal Growth Factor; ErbB Receptors; Humans; Iodine Radioisotopes; Osteosarcoma; Parathyroid Hormone; Tetradecanoylphorbol Acetate

The growth of MG63 human osteosarcoma cell line in 5% serum is stimulated by epidermal growth factor (EGF), platelet-derived growth factor (PDGF), or heparin-binding growth factor-1 (HBGF-1). The mitogenic effect of EGF and PDGF is completely blocked by TFG-beta at 1 ng per ml and the effect of HBGF-1 is attenuated by 75-80%. Treatment of MG63 cells with TGF-beta reduces HBGF-1 receptor binding affinity from 1.24 x 10(-11) M to 3.51 x 10(-11) M with no change on the receptor number (1.1 x 10(3) per cell). The receptor-binding affinity of EGF and PDGF is not altered by TGF-beta treatment; however, the number of EGF receptor is increased by 25%. Both EGF and PDGF stimulate MG63 cellular tyrosine kinase activity, and such stimulation is inhibited by TGF-beta pretreatment. No change in the cellular protein tyrosine phosphorylation pattern can be detected in HBGF-1-stimulated cells with and without TGF-beta pretreatment. These data suggest that TGF-beta inhibits EGF and PDGF mitogenicity by blocking EGF- and PDGF-stimulated tyrosine kinase activity and attenuates HBGF-1 mitogenicity by decreasing its receptor affinity.

Topics: Cell Division; Cell Line; Cell Membrane; Epidermal Growth Factor; ErbB Receptors; Fibroblast Growth Factor 1; Growth Substances; Heparin; Humans; Kinetics; Mitogens; Molecular Weight; Osteosarcoma; Phosphoproteins; Platelet-Derived Growth Factor; Protein-Tyrosine Kinases; Receptors, Cell Surface; Receptors, Mitogen; Receptors, Platelet-Derived Growth Factor; Receptors, Vascular Endothelial Growth Factor; Transforming Growth Factors; Tumor Cells, Cultured

The SK-Luci-6 cell line, established from a large-cell anaplastic lung tumor of a patient with humoral hypercalcemia of malignancy (HHM), was investigated to identify osteolytic factors produced that might mediate HHM. Most HHM-associated tumors are thought to produce parathyroid hormone-related proteins or transforming growth factor (TGF) alpha. SK-Luci-6 cells formed s.c. tumors and induced hypercalcemia in athymic nude mice. Serum-free conditioned medium from SK-Luci-6 cultures induced bone resorption in neonatal mouse calvariae in vitro, and also contained TGF-beta activity and mitogenic activity. SK-Luci-6 cell conditioned medium did not displace [125I]epidermal growth factor binding to cell receptors or stimulate cyclic AMP formation in rat osteosarcoma cells, suggesting that the conditioned medium did not contain TGF-alpha or parathyroid hormone-related proteins. The osteolytic, TGF-beta, and mitogenic activities copurified in several chromatographic separations: gel filtration in acid and then in guanidine HCl; ion exchange; and reverse phase. The results suggest that in the HHM-associated SK-Luci-6 tumor, the causative osteolytic factor produced by the tumor cells is not a parathyroid hormone-related protein or TGF-alpha but, rather, may be a TGF-beta.

Topics: Animals; Calcium; Cell Line; Chick Embryo; Cyclic AMP; Epidermal Growth Factor; Female; Humans; Hypercalcemia; Lung Neoplasms; Male; Mice; Mice, Nude; Osteolysis; Osteosarcoma; Transforming Growth Factors

A clonal cell line (Saos-2/B-10) derived from human osteosarcoma Saos-2 cells had the same osteoblastic characteristics as the mother line, but lacked sensitivity to parathyroid hormone (PTH) at early passages. At later passages (greater than 70) the cells became very sensitive to PTH (0.1 nmol/l). The absence of PTH-stimulatable adenylate cyclase correlated with the secretion of an adenylate cyclase-stimulatory activity which had the properties of the recently characterized PTH-like peptide (PTH-LP). This activity was inhibited by the PTH antagonist [8norleucyl,18norleucyl,34tyrosinyl]bovine PTH-(3-34)amide and could be neutralized by an antiserum raised against the synthetic PTH-LP-(1-34). Hybridization with a human PTH-LP cDNA showed that these cells produce two PTH-LP mRNAs of approximately 1.5 and 1.8 kb. The production of PTH-LP was stimulated by 12-O-tetradecanoyl-phorbol-13-acetate (TPA; 150 nmol/l) and epidermal growth factor (EGF; 10 ng/ml). The increased accumulation of PTH-LP in conditioned media in response to TPA was seen after 1 h and levelled off at 6 h. In contrast, EGF stimulation was lower at 3 and 6 h but continued for 24 h. Both agents increased PTH-LP mRNA levels in Saos-2/B-10 cells. A TPA analogue which does not stimulate protein kinase C had no effect on PTH-LP production. Cycloheximide blocked the stimulatory effect of both TPA and EGF and the TPA effect was blocked by actinomycin D, suggesting transcriptional control. The regulation of PTH-LP by these agents may offer clues regarding the association of this protein with malignancy.

Topics: Adenylyl Cyclases; Cell Line; Epidermal Growth Factor; Humans; Neoplasm Proteins; Osteosarcoma; Parathyroid Hormone; Parathyroid Hormone-Related Protein; RNA, Messenger; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

The objective of this study was to establish whether human recombinant tumor necrosis factor (TNF) can significantly stimulate the proliferation of some tumor cells. Treatment with TNF had little or no effect on the growth of human tumor cells and murine NIH/3T3 cells cultured in medium with high serum concentration. Two tumor lines, SK-MEL-109 melanoma and HOS osteosarcoma cells, were adapted to grow in medium supplemented with 0.5% serum. The growth of these SK-MEL-109 cells was inhibited by TNF, but that of the HOS cells was greatly stimulated by TNF in a dose-dependent way. Treatment with 10 ng/ml of TNF resulted in a two-fold increase in the rate of cell division. This effect of TNF was also shown by measuring DNA and protein synthesis. The continuous presence of TNF was not required for its mitogenic activity on HOS cells cultured with 0.5% serum, since treatment for only one day with TNF resulted in prolonged growth stimulation. The failure of TNF to promote division of cells cultured in medium with 10% serum may possibly be explained by the presence of saturating amounts of growth factors in serum. Interferons abolished the mitogenic activity of TNF on HOS cells. Furthermore, TNF did not show synergism with insulin or epidermal growth factor in stimulating growth of these cells. The level of c-myc mRNA was increased five-fold after 30 minutes of treatment with TNF. This shows that TNF is a growth factor for HOS cells and that it induces accumulation of c-myc mRNA.

Topics: Animals; Cell Division; Culture Media; DNA; Epidermal Growth Factor; Humans; Insulin; Interferons; Mitosis; Nucleic Acid Hybridization; Osteosarcoma; Protein Biosynthesis; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-myc; Proto-Oncogenes; RNA, Messenger; Transcription, Genetic; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Collagenases that specifically cleave native collagen at neutral pH have been implicated in the maintenance and turnover of connective tissue. In bone, the origin of neutral collagenase has remained equivocal, although recent studies have indicated that it is synthesized by the osteoblast. In the present work, regulation of secretion of neutral collagenase and a collagenase inhibitory activity was investigated using the osteoblastic tumor cell line UMR 106-01 and a variety of bone-resorbing agents. Under basal conditions, UMR 106-01 cells produced very low levels of collagenase but substantial amounts of the inhibitory activity. Exposure to PTH and, to a lesser extent, 1,25-dihydroxyvitamin D3, prostaglandin E2, retinoic acid, and epidermal growth factor stimulated the release of collagenase, an effect not seen with interleukin-1 or heparin. The stimulation of collagenase by PTH was dose dependent, with a half-maximal response occurring at 10(-8) M. Inclusion of isobutylmethylxanthine decreased the concentration of PTH required to produce half-maximal stimulation to 2 X 10(-10) M, indicating action via cAMP. With respect to the inhibitory activity, PTH and epidermal growth factor were the only agents, among those tested, able to enhance its production. Both hormones caused a 50-100% increase over control levels 72 h after hormone administration. There were notable differences in the time courses of production of collagenase and the inhibitor. After treatment with PTH, the enzyme reached maximal concentrations between 12-48 h, but declined to undetectable levels by 96 h. In contrast, the inhibitory activity was secreted in a linear fashion, with the highest concentrations achieved around 72-96 h. These results suggest a complex pattern of regulation of collagenase and inhibitor secretion by the osteoblastic cell, with the steady accumulation of inhibitor perhaps being responsible for the ultimate curtailment of enzyme activity.

Topics: Animals; Bone Resorption; Calcitriol; Cell Line; Cyclic AMP; Dinoprostone; Epidermal Growth Factor; Female; Kinetics; Microbial Collagenase; Osteosarcoma; Parathyroid Hormone; Prostaglandins E; Rats; Tretinoin; Uterus

The effects of transforming growth factor alpha (TGF-alpha) and epidermal growth factor (EGF) on parathyroid hormone (PTH)-responsive adenylate cyclase were examined in clonal rat osteosarcoma cells (UMR-106) with the osteoblast phenotype. Recombinant TFG-alpha and EGF incubated with UMR-106 cells for 48 h each produced concentration-dependent inhibition of PTH-responsive adenylate cyclase, with maximal inhibition of 38-44% at 1-3 ng/ml of either growth factor. TGF-alpha and EGF also inhibited beta-adrenergic agonist (isoproterenol)-stimulated adenylate cyclase by 32%, but neither growth factor affected enzyme response to prostaglandin or basal (unstimulated) activity. Nonreceptor-mediated activation of adenylate cyclase by forskolin and cholera toxin was inhibited 18-20% by TGF-alpha and EGF. Pertussis toxin augmented PTH-stimulated adenylate cyclase, suggesting modulation of PTH response by a functional inhibitory guanine nucleotide-binding regulatory component of the enzyme. However, pertussis toxin had no effect on TGF-alpha inhibition of PTH response. Growth factor inhibition of PTH response was time-dependent, with maximal inhibition by 4-12 h of TGF-alpha exposure, and was reduced by prior treatment of UMR-106 cells with cycloheximide. TGF-alpha was not mitogenic for UMR-106 cells. The results indicate that TGF-alpha and EGF selectively impair PTH- and beta-adrenergic agonist-responsive adenylate cyclase of osteoblast-like cells. Growth factor inhibition of adenylate cyclase may be exerted at the receptor for stimulatory agonist and at nonreceptor components excluding pertussis toxin-sensitive guanine nucleotide-binding regulatory proteins. The inhibitory action of growth factors may also require protein synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenylyl Cyclase Inhibitors; Animals; Cell Line; Clone Cells; Epidermal Growth Factor; Kinetics; Osteoblasts; Osteosarcoma; Parathyroid Hormone; Peptides; Rats; Recombinant Proteins; Transforming Growth Factors

Influx of extracellular Ca++ into bone cells has been postulated as an early action of PTH and other bone resorption-stimulating factors. To test this hypothesis directly, we measured the cytosolic free Ca2+ concentration ([Ca2+]i) in two hormone-responsive human (SaOS-2 and G-292) and two rat osteosarcoma cell lines (Ros 25/1 and Ros 17/2.8) and in primary cultures of bone cells from neonatal mouse calvaria using the fluorescent Ca2+ indicator Quin 2. Actions of bovine PTH-(1-34), vasoactive intestinal peptide, epidermal growth factor, prostaglandin E2, and ionomycin were studied. Medium cAMP (20 min; 37 C; 25 microM 3-isobutyl-1-methylxanthine) was quantitated by RIA. Basal [Ca2+]i was: SaOS-2, 126 +/- 8 nM; G-292, 61 +/- 6 nM; Ros 25/1, 109 +/- 15 nM; Ros 17/2.8, 363 +/- 42 nM; and primary cultures, 266 +/- 39 nM (mean +/- SE; n = 3-14). In each cell type, no acute (1 sec to 20 min) spike in [Ca2+]i was observed in response to PTH (24-120 nM), vasoactive intestinal peptide (100 nM), epidermal growth factor (17 nM), or prostaglandin E2 (2.8 microM). However, in SaOS-2 cells only, PTH reproducibly increased [Ca2+]i 10-15% above basal values beginning about 3 min after hormone addition, and this small increase returned to baseline at 15-20 min. Ionomycin (100 nM) elicited an immediate spike in [Ca2+]i to levels 2- to 4-fold above basal in all cells; the peak [Ca2+]i decayed rapidly (within 4-5 min) to baseline in G-292, Ros 25/1, and Ros 17/2.8 cells. The decay of peak [Ca2+]i in SaOS-2 was prolonged. To test for intact hormone responses in Quin 2-loaded cells, cAMP accumulation was measured. In SaOS-2 and Ros 17/2.8, both control and Quin 2-loaded cells showed similar increases in cAMP in response to PTH. Considering the limitations of the Quin 2 technique, we conclude that in the four hormone-responsive bone cell lines and primary cultures of bone cells tested, acute elevation of [Ca2+]i is not an inevitable consequence of receptor occupancy and/or adenylate cyclase activation by bone resorption-stimulating hormones.

Topics: Aminoquinolines; Animals; Bone Resorption; Calcium; Cell Line; Cells, Cultured; Cyclic AMP; Cytosol; Dinoprostone; Epidermal Growth Factor; Ethers; Humans; Ionomycin; Membrane Potentials; Mice; Osteosarcoma; Parathyroid Hormone; Prostaglandins E; Rats; Vasoactive Intestinal Peptide

The epidermal growth factor receptor in cells of the UMR 106-06 clonal osteoblast line has been shown to be structurally similar to that previously characterized in other cell lines. A specific receptor component of approximately 165,000-185,000 Mr has been identified by polyacrylamide gel electrophoresis using the chemical crosslinker disuccinimidyl suberate to crosslink 125I-EGF to its receptor. Tunicamycin treatment of cells resulted in a dose-dependent loss of binding suggesting involvement of glycosyl moieties in EGF binding to its receptor. Competitive binding studies carried out using wheat germ lectin (WGL), concanavalin A (CON.A.), soybean lectin (SBL), and lentil lectin (ILL) to compete for binding of 125I-EGF revealed that CON A, WGL, and to a lesser extent LL could inhibit EGF binding; SBL was without effect. Treatment of the cells with neuraminidase which cleaves terminal sialic acid residues resulted in total loss of binding while alpha-glucosidase, beta-N-acetylglucosaminidase and alpha-mannosidase were without effect. These data indicate a specific interaction of EGF with terminal sialic acid residues of the EGF receptor. However, it would seem that the mannose residues which appeared to modify EGF binding were not available for the action of the above enzymes due to the presence of sialic acid.

Topics: Animals; Binding Sites; Binding, Competitive; Bone and Bones; Clone Cells; Epidermal Growth Factor; ErbB Receptors; Kinetics; Lectins; Molecular Weight; N-Acetylneuraminic Acid; Osteosarcoma; Rats; Receptors, Cell Surface; Sialic Acids

Human recombinant transforming growth factor alpha (TGF alpha), which binds to the epidermal growth factor (EGF) receptor and causes several biological effects similar to those caused by EGF, was compared with murine EGF for its effects on a number of parameters of bone cell metabolism. TGF alpha stimulated bone resorption in two organ culture systems, the fetal rat long bone and neonatal mouse calvarial systems. TGF alpha stimulated bone resorption at concentrations as low as 0.1 ng/ml. TGF alpha effects on bone resorption in mouse calvariae were inhibited by indomethacin, suggesting that, like EGF, its effects were mediated by prostaglandin synthesis. TGF alpha had a different time course of action on bone resorption from that of EGF, causing more rapid release of previously incorporated 45Ca from bone cultures, suggesting that TGF alpha does not function on bone as a simple EGF analogue. TGF alpha also caused effects on osteoblast function resembling those of EGF. It inhibited alkaline phosphatase activity in cultured rat osteosarcoma cells with the osteoblast phenotype and inhibited collagen synthesis in fetal rat calvaria at concentrations of 1.0 ng/ml. The lowest concentration of TGF alpha (expressed as nanogram equivalents of EGF per ml) required to produce a response in all of the systems tested was about 1/10th of that needed for EGF to produce a similar effect. These results indicate that TGF alpha is a potent stimulator of bone resorption and inhibitor of bone formation as assessed by inhibition of collagen synthesis and alkaline phosphatase activity and are consistent with the hypothesis that TGF alpha may be responsible, at least in part, for the bone resorption associated with some tumors.

Topics: Alkaline Phosphatase; Animals; Bone Resorption; Collagen; Dose-Response Relationship, Drug; Epidermal Growth Factor; Indomethacin; Osteosarcoma; Peptides; Rats; Recombinant Proteins; Time Factors; Transforming Growth Factors

The plasminogen activator (PA) activity of clonal rat osteogenic sarcoma cell (phenotypically osteoblast) and of osteoblast-rich rat calvarial cells is shown to be increased by treatment with the bone-resorbing hormones, PTH, 1,25-dihydroxyvitamin D3, prostaglandin E2, and epidermal growth factor. Dose-dependent increases were observed, after a lag period of 4 to 8 h. Stimulated and control PA activities were inhibited by actinomycin D and cycloheximide but not by cytosine arabinoside. Glucocorticoid hormones prevented the hormone stimulation, but other steroids did not. Calcitonin had no effect either on basal or on hormone-treated PA activity. Isobutyl-methylxanthine alone increased PA activity and enhanced responsiveness to PTH and to prostaglandin E2. These data point to a common pathway in the actions upon osteoblasts of several hormones with diverse initial cellular actions and raise the possibility that the PA/plasmin system may contribute to cellular mechanisms of bone turnover.

Topics: Animals; Bone Resorption; Calcitonin; Calcitriol; Dinoprostone; Epidermal Growth Factor; Glucocorticoids; Kinetics; Microbial Collagenase; Nucleotides, Cyclic; Osteoblasts; Osteosarcoma; Parathyroid Hormone; Plasminogen Activators; Prostaglandins E; Protein Biosynthesis; Rats; RNA; Serum Albumin, Bovine

U-2 OS human osteosarcoma cells synthesize, process and secrete a platelet-derived growth factor (PDGF)-like mitogen. Incubation of these cells with 1 mM suramin unmasks PDGF receptor sites which are normally occupied or down regulated by the secreted endogenous PDGF-like mitogen. Partially purified preparations of metabolically labelled U-2 OS conditioned medium binds to U-2 OS cells and binding is inhibited by excess PDGF. These findings suggest that U-2 OS cells are capable of autocrine stimulation.

Topics: Binding, Competitive; Cell Line; Epidermal Growth Factor; Humans; Kinetics; Osteosarcoma; Platelet-Derived Growth Factor; Receptors, Cell Surface; Receptors, Platelet-Derived Growth Factor; Suramin

The FM-2 cell line is a cloned, immortalized cell line derived from a human osteosarcoma. Conditioned medium from FM-2 cultures contains a factor which stimulates calcium mobilization from fetal rat bone organ cultures. Treated bones contain increased numbers of osteoclasts and decreased bone matrix. This factor has a molecular weight of approximately 29,000 as determined by gel filtration. Its biological activity is dependent on a protein moiety and is completely inhibited by calcitonin. Its synthesis by the FM-2 line is dependent on cell density and replenishment of fresh medium. This factor is not parathyroid hormone, a vitamin D metabolite, prostaglandin E, epidermal growth factor, or osteoclast-activating factor, all of which have bone-resorbing activities. Also, FM-2-conditioned medium inhibits collagen synthesis in fetal rat calvaria cells and decreases alkaline phosphatase levels in an osteoblastic cell line, and these two properties coelute with the calcium-mobilizing factor from a hydroxylapatite column. These biological products, synthesized by a cell line derived from a tumor, may represent physiological factors normally synthesized by a subpopulation of bone cells.

Topics: Animals; Biological Assay; Biological Products; Bone and Bones; Calcium; Cell Line; Cells, Cultured; Clone Cells; Cytokines; Epidermal Growth Factor; Female; Fetus; Humans; Mice; Osteosarcoma; Pregnancy

Hormonal control of plasminogen activator (PA) was studied in clonal rat osteogenic sarcoma cells which are phenotypically osteoblast, and in osteoblast-rich rat bone cell cultures. The bone-resorbing hormones (parathyroid hormone, prostaglandin E2, epidermal growth factor and 1,25-dihydroxyvitamin D3) stimulated PA activity in both cell types. The relative efficacies of vitamin D metabolites and of prostanoids reflect their relative potencies as stimulators of bone resorption.

Topics: Animals; Bone Resorption; Calcitriol; Cell Line; Clone Cells; Dinoprostone; Epidermal Growth Factor; Osteoblasts; Osteosarcoma; Parathyroid Hormone; Plasminogen Activators; Prostaglandins E; Rats; Skull

The phorbol ester analog, mezerein, is a weak complete and Stage 1 tumor promoter; however, it is as potent as the most active phorbol esters as a second stage promoter and inflammatory agent. Therefore, mezerein is a useful compound for studying responses associated with Stage 1 or Stage 2 promotion. In this paper, we show that in G-292 osteosarcoma cells in culture, mezerein is 25-fold more potent in causing a decrease in binding of epidermal growth factor to its specific cellular receptor than in inducing prostaglandin E2 production. This differential potency for these two actions was not noted for other phorbol esters. Our findings indicate that mezerein interacts with the major phorbol dibutyrate receptor to increase prostaglandin E2 production and also either with a distinct cellular target with a higher affinity or the same target with increased efficacy to cause a decrease in the binding of epidermal growth factor. These human osteosarcoma cells thus provide a model system to facilitate analysis of phorbol ester receptor heterogeneity.

Topics: Binding, Competitive; Caenorhabditis elegans Proteins; Carrier Proteins; Dinoprostone; Diterpenes; Epidermal Growth Factor; Humans; Kinetics; Osteosarcoma; Phorbol 12,13-Dibutyrate; Phorbol Esters; Phorbols; Prostaglandins E; Protein Kinase C; Receptors, Drug; Terpenes; Tetradecanoylphorbol Acetate

Normal and malignant osteoblast-like cells in culture have been shown to possess specific, high affinity receptors for epidermal growth factor (EGF). In this study, the mitogenic response to EGF was examined in a clonal line of a rat osteogenic sarcoma (UMR 106) and in osteoblast-rich newborn rat calvarial cells. Twenty-four hour treatment of UMR 106 cells with EGF in doses ranging from 10(-12) M to 2 X 10(-8) M stimulated the incorporation of [3H]thymidine and DNA synthesis in a dose-dependent manner. This short-term stimulatory effect was sustained in long-term culture with a dose-dependent increase in cell proliferation by calvarial cells. A lag period of 8 h occurred before significant stimulation of [3H]thymidine incorporation was observed. Commitment to increased incorporation of [3H]thymidine required a minimum of 6 h continuous incubation with EGF. These results establish the osteoblast as a target cell for EGF action on bone.

Topics: Animals; Cell Division; Cell Line; DNA; Dose-Response Relationship, Drug; Epidermal Growth Factor; Osteoblasts; Osteosarcoma; Rats; Skull

Human platelet-derived growth factor (PDGF) stimulated the production of prostaglandin E2 (PGE2) by G-292 cells, a clonal line of human osteosarcoma cells. Half-maximal stimulation occurred with 9 ng/ml PDGF and maximal stimulation, 3-fold above control values, occurred with 40 ng/ml of the protein. Treatment of G-292 cells with 40 ng/ml PDGF also reduced the binding of iodinated epidermal growth factor (EGF) to the EGF receptor on G-292 cells. The effect was time-dependent, and EGF binding was reduced to 60% of control by 24-48 h. PDGF did not, however, compete directly for binding to the EGF receptor. The effects of PDGF and EGF on increased PGE2 production appeared to be additive at all concentrations tested, indicating that they may act through a common pathway, but not via the same membrane receptors.

Topics: Cells, Cultured; Epidermal Growth Factor; ErbB Receptors; Growth Substances; Humans; Osteosarcoma; Peptides; Platelet-Derived Growth Factor; Prostaglandins; Receptors, Cell Surface

Topics: Calcium; Cell Line; Dinoprostone; Epidermal Growth Factor; ErbB Receptors; Humans; Osteosarcoma; Phorbol Esters; Phorbols; Prostaglandins E; Receptors, Cell Surface; Tetradecanoylphorbol Acetate

Topics: Animals; Antibodies; Blood Platelets; Cross Reactions; Epidermal Growth Factor; Epitopes; Fibroblast Growth Factors; Growth Substances; Humans; Immunosorbent Techniques; Osteosarcoma; Peptides; Platelet-Derived Growth Factor; Rabbits; Radioimmunoassay

Topics: Cyclic AMP; Dinoprost; Dinoprostone; DNA; Epidermal Growth Factor; ErbB Receptors; Humans; Kinetics; Leupeptins; Osteosarcoma; Parathyroid Hormone; Prostaglandins E; Prostaglandins F; Receptors, Cell Surface; Time Factors